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EP2949350B1 - Réseau de tissus biologiques creux artificiel et procédé de préparation associé - Google Patents

Réseau de tissus biologiques creux artificiel et procédé de préparation associé Download PDF

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EP2949350B1
EP2949350B1 EP14170465.0A EP14170465A EP2949350B1 EP 2949350 B1 EP2949350 B1 EP 2949350B1 EP 14170465 A EP14170465 A EP 14170465A EP 2949350 B1 EP2949350 B1 EP 2949350B1
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network
curve
section
section curve
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EP2949350A1 (fr
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Bahattin Koc
Can Kücukgül
Saime Burce Özler
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Sabanci Universitesi
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Sabanci Universitesi
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    • G—PHYSICS
    • G05—CONTROLLING; REGULATING
    • G05B—CONTROL OR REGULATING SYSTEMS IN GENERAL; FUNCTIONAL ELEMENTS OF SUCH SYSTEMS; MONITORING OR TESTING ARRANGEMENTS FOR SUCH SYSTEMS OR ELEMENTS
    • G05B17/00—Systems involving the use of models or simulators of said systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06—Blood vessels
    • A61F2/062—Apparatus for the production of blood vessels made from natural tissue or with layers of living cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/50—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for simulation or modelling of medical disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06—Blood vessels
    • A61F2002/065—Y-shaped blood vessels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Definitions

  • the present invention relates to artificial biological tissue networks and a method for preparation thereof.
  • Cardiovascular disease is the leading cause of deaths worldwide.
  • the most common treatments for cardiovascular health diseases are autograft and blood vessel transplantation which has limitations due to lack of donors and the patients' conditions may not allow harvesting one. Additionally, extracting of an autograft may not be possible if all the possible grafts are extracted from the harvested site or the disease has already been advanced.
  • tissue engineering is to provide a possible alternative for such grafts. Recently, tissue engineering and regenerative medicine aim to provide alternative treatments and fast recovery for the patients suffering from cardiovascular diseases. (doi: 10.1155/2012/956345)
  • tissue engineering strategies are based on the cell seeding into synthetic, biological or composite scaffolds providing a suitable environment for cell attachment, proliferation and differentiation.
  • Cells are seeded into synthetic, biological or composite scaffolds which supply a suitable environment for cell attachment, proliferation and differentiation and have the same functional role as an extracellular matrix (ECM) until the cells create their own ECM.
  • ECM extracellular matrix
  • the seeded scaffolds' degradation could cause immunogenic and unforeseen side effects after in-vivo implantation.
  • the scaffolds have been fabricated with controlled internal architecture using 3D printing techniques (doi: 10.1088/1758-5082/3/3/034106, doi: 10.1016/j.cad.2013.07.003).
  • 3D scaffolds are designed to act as an artificial ECM until the cells form their own ECM, it is challenging to fabricate a controlled porous structure with a desired internal architecture repetitively.
  • functional vascularization of 3D scaffolds is compulsory needed for nutrition and oxygen supply to the engineered tissue.
  • endothelial cells or scaffolds are developed (doi: 10.1016/j.addr.2011.03.004).
  • shape and orientation of branches are limited to be parallel with the flat surface on which the cell paste pieces of living cells and their support material pieces are laid. This is a highly binding limitation, which does not fit the natural organization of cells and the shapes of real blood vessels, which have generally uneven shapes and branch orientations.
  • the longitudinal multicellular aggregate preparation method explained in said document requires several repetitive manual bioink preparation steps of multicellular aggregates into/from capillaries; which have to be performed with extreme precision; hence, the reproducibility and speed of said steps can be considered as low. Therefore an alternative method replacing said steps, thus minimizing the human intervention and maximizing the reproducibility is extremely important for bioprinting of said networks.
  • Multicellular spherical and cylindrical aggregates have been fabricated with 3D printing methods. Flexibility in tube diameter and wall thickness is obtained and most significantly branched macrovascular structures are constructed with this method (doi:10.1088/1758-5082/4/2/022001).
  • human embryonic stem cell spheroid aggregates are formed with a valve-based cell printer and they have controllable and repeatable sizes. This work shows that the printed cells are mostly viable and have the potential to differentiate into any of the three germ layers (pluripotency) (doi:10.1088/1758-5082/5/1/015013).
  • Valve scaffold tissue engineering has the potential for fabricating blood vessels e.g. aortic valve hydrogel scaffolds that can grow, remodel and integrate with the patient.
  • aortic valve hydrogel scaffolds that can grow, remodel and integrate with the patient.
  • root wall and tri-leaflets are 3D printed with poly-ethylene-glycol-diacrylate (PEG-DA) hydrogels.
  • PEG-DA poly-ethylene-glycol-diacrylate
  • Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds maintained near 100% viability over 21 days (doi: 10.1088/1758-5082/4/3/035005).
  • Primary object of the present invention is to eliminate the above-mentioned shortcomings in the prior art.
  • Another object of the present invention is to provide a self-supporting scaffold-free artificial hollow biological tissue network for replacement of living tissue.
  • an object of the present invention is to provide an artificial biological tissue network with a high reproducibility and without requiring any manual intervention.
  • Still further an object of the present invention is to provide an artificial biological tissue network which is constructed in bottom-up direction.
  • Yet another object of the present invention is to provide an artificial biological tissue network which achieves a natural mechanical strength in a shorter time with respect to the prior art.
  • Another object of the present invention is to provide a method for obtaining such artificial biological tissue network directly based on medical images of the targeted tissue or organ.
  • a further object of the present invention is to provide a branched self-supporting scaffold-free artificial hollow biological tissue network for replacement of living tissue.
  • a network for replacement of a living tissue said network is a scaffold-free artificial hollow biological tissue network as dislcosed in claim 1 comprising a plurality of longitudinal multicellular aggregates arranged in a plurality of bioprinted layers which are located on top of one another, further comprising an inner surface and an outer surface, at least one of said bioprinted layers is in shape of a planar closed loop such that a conduit for conveying fluids is defined, and said longitudinal multicellular aggregate is a mixture of at least two cell types. Also a method for biomodeling and planning said network is proposed.
  • the present invention proposes a self-supporting scaffold-free artificial hollow biological tissue network (also named as 'network' hereafter) explained in Claim 1, which overcomes the above-mentioned shortcomings of the prior art.
  • Said network (100) is shaped as a self-supporting three-dimensional (3D) bioprinted structure which is built from continuous longitudinal (e.g. cylindrical) multicellular aggregates of living cells, without employing any non-biological material e.g. scaffold remaining in contact with said network after implantation to a patient.
  • the term 'self-supporting' here means that the network (100) is supported by a removable plurality of support structures (12) as explained in this text, until the completion of the maturation process of said network, and after removal of support structures (12) the network (100) mainly comprises living cells fused and adhered in accordance with their natural abilities, which do not further require to be supported by any unnatural means e.g. scaffold.
  • the term 'multicellular' means that said aggregate comprises a plurality of living cells. Additionally, said aggregate is 'heterocellular' since it comprises a mixture comprising at least two cell types selected from a group comprising following cell types: fibroblasts, endothelial cells, smooth muscle and stem cells.
  • a plurality of said longitudinal multicellular aggregates (11) are arranged in a plurality of bioprinted layers (22) which are located on top of one another, and at least one of said longitudinal multicellular aggregate layers is in shape of a planar closed loop such that a conduit for conveying fluids is defined.
  • said network (100) is a branched conduit, e.g. a branched blood vessel as shown in Figure 6 (b-d).
  • branched conduit comprises at least two branches (19) converged on at least one sector of said network.
  • Each closed loop of said longitudinal multicellular aggregate (11) can preferably be in contact with a support structure (12), which is biocompatible, bio-inert and also in form of longitudinal pieces, said contact is at both an inner surface (20) and an outer surface (21) of the network (100) for serving as a mold during a maturation process of the network.
  • Said support structure (12) e.g. hydrogel
  • Said support structure (12) can preferably comprise additional synthetic or biological substances for providing support to the viability of cells during the maturation of said network. Suitable substances to this end are known and available for a skilled person in the art.
  • longitudinal multicellular aggregate (11) Longitudinal geometry of said longitudinal multicellular aggregate (11) accelerates the maturation process of said network (100) since it facilitates perfusion between said cells in comparison with that between the cells in known spherical aggregates, thus the self-assembly, connection and partially cohesion of said cells is improved thanks to increased contact between adjacent bioprinted layers (22).
  • cylindrical longitudinal multicellular aggregate with a closed-loop cross-section was chosen as said longitudinal geometry.
  • Said closed-loop cross-section can have disk geometry as clearly shown in Figure 5 (a)
  • the longitudinal multicellular aggregate (11) can also have elliptical or other suitable closed-loop cross-section geometries besides of disk.
  • Each longitudinal multicellular aggregate (11) can preferably be in direct contact with at least one adjacent longitudinal multicellular aggregate (11) providing a cell-to-cell contact between said layers.
  • the longitudinal multicellular aggregates (11) in a bioprinted network (100) according to the present invention can be completely or partially cohered with each other.
  • Said longitudinal multicellular aggregate (11) has a length/diameter aspect ratio in the range of 20 to 250. Additionally, said longitudinal multicellular aggregate has a diameter within the range of 100 to 2500 micrometers.
  • a 3D printer was used for depositing longitudinal multicellular aggregate (11) and the support structure (12) with its multiple deposition heads having capillaries.
  • the capillaries for bearing and placing longitudinal multicellular aggregate (11) and support structure (12) can be made of glass or any other suitable bio-inert material e.g. steel or bio-inert polymers; and have inner diameters within the range of 100 to 2500 micrometers.
  • the diameters of both capillaries for longitudinal multicellular aggregate (11) and support structure (12) were of about 500 ⁇ m.
  • a 3D bioprinter can then be controlled directly by scripts generated by the methodology according to the present invention, and it can print longitudinal multicellular aggregates (11) and support structures (12) layer by layer to form desired tissue structures i.e. networks (100).
  • a biocompatible, bio-inert, thermo-reversible hydrogel e.g. agarose-based hydrogels can be used as support structure (12) for 3D printing of said network.
  • NovoGel As the support structure (12), NovoGel (a commercial product by Organovo) was used for the experimental work explained below.
  • NovoGel 2% (w/v) with phosphate buffered saline (PBS: Hyclone 1X by Thermo Scientific) with Ca 2+ and Mg 2+ salts solution was prepared by autoclaving it in accordance with standard liquid sterilization procedures.
  • PBS phosphate buffered saline
  • Ca 2+ and Mg 2+ salts solution was prepared by autoclaving it in accordance with standard liquid sterilization procedures.
  • 2% agarose solution with PBS was prepared first. After a sterilization process, 20 mL agarose solution is transferred into a petri dish bottom covering the entire dish bottom surface.
  • the following sequential steps are preferably applied at least once between said steps 'a' and 'b' in order to allow said cells to aggregate and initiate cell-cell adhesions,
  • an automated bioprinter having said capillary can preferably be employed.
  • Employing an automated bioprinter in connection with said capillary even further reduces the human intervention at forming said longitudinal heterocellular aggregate (11) and even further increases the reproducibility.
  • Said automated bioprinter working according to the method of the present invention arranges in-capillary-formation of longitudinal multicellular aggregates (11) and support structures (12) having respective calculated lengths for each bioprinted layer (22), such that non-interrupted closed loops of longitudinal multicellular aggregates (11) and support structures (12) are formed.
  • said aspiration is performed using an automated bioprinter comprising said capillary, and said part fills a predetermined length of said capillary, which length is equal to a predetermined length of a longitudinal multicellular aggregate (11).
  • Such longitudinal multicellular aggregate (11) is suitable for use in obtainment of self supporting scaffold-free artificial biological tissue networks (100) according to the present invention.
  • Said method is employed in an example experiment according to the present invention as follows: In this exemplary experiment, a mixture comprising smooth muscle cells, endothelial cells and early passage human dermal fibroblast (HDF) cells were used. The percentage of each cell type in said heterocellular mixture is preferably decided based on the tissue type. In this exemplary experiment, 25% endothelial cells are mixed with 75% HDF cells to form heterocellular mixture.
  • HDF human dermal fibroblast
  • the cells were cultured at 37°C and under 5% CO 2 using Dulbecco's Modified Eagle's Medium (DMEM, provided by Sigma, Germany) supplemented with 10% fetal bovine serum (FBS, provided by Sigma, Germany), 1% Penicillin-Streptomycin (provided by Sigma, Germany).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • Penicillin-Streptomycin provided by Sigma, Germany
  • tissue culture flasks were cultured in tissue culture flasks. Following the removal of the culture medium from culture flasks, cells were washed with PBS. Said cells can be detached from the tissue culture flasks using two alternative ways: they can be either detached by incubating with trypsin solution from porcine pancreas (provided by Sigma, Germany) (the trypsin concentration was 1X and the contact time was 10 minutes), or by using cell scrapers. Following the detachment step, remaining trypsin was rinsed/neutralized using serum containing medium, and the cells were centrifuged at 200 x g for 5 minutes, then supernatant was discarded thus a cell pellet is obtained.
  • T25 tissue culture flasks TPP, Germany
  • T25 tissue culture flasks TPP, Germany
  • T25 tissue culture flasks were incubated at 37°C and 5% CO 2 on an orbital shaker and shaken at 160 rpm for 60 minutes.
  • the cells were collected into 15 mL sterile falcon tubes and the collected cell suspensions were centrifuged at 200 x g for 5 minutes.
  • the cell pellet was resuspended (for this experiment, in a 1 mL medium) and transferred into Eppendorf tubes (for this experiment, 1.5 ml) and centrifuged again at 1000 x g for 4 minutes to form a dense cell pellet.
  • cell pellets in the Eppendorf tubes were non-formed multicellular aggregates (pellets) to be aspired into capillaries for formation of longitudinal multicellular aggregates (11) for continuous bioprinting automatically according to the said developed algorithms.
  • said aspiration and bioprinting are performed automatically by an automated bioprinter having a capillary(ies).
  • the bioprinted constructs were incubated for 1-7 days so that the longitudinal multicellular aggregates (11) fuse together, and an artificial biological tissue network (100) is formed.
  • the formed biological tissue network is preferably put in a bioreactor where a flow of medium pass through to further maturate the fused network (100) until the replacement of a targeted tissue is constituted.
  • FIG. 6 (a-d) shows several schematic examples for artificial biological tissue networks (100) according to the present invention, which are of various levels of shape complexities.
  • Longitudinal multicellular aggregates (11) obtained with the method according to the present invention are free of any coating.
  • each bioprinted layer (22) of longitudinal multicellular aggregates (11) will be in a direct contact with adjacent bioprinted layers (22) free of any mass transfer barrier.
  • This facilitates said bioprinted layers (22) to fuse together and contract, so that the constitution of a network (100) with a natural strength occurs in a shortened time in comparison with prior art methods, since the cells in adjacent bioprinted layers (22) are in contact with each other throughout the bioprinted network (100).
  • the continuous longitudinal geometry of the longitudinal multicellular aggregate (11) provides a high number of neighboring cells in each layer, which further facilitates the constitution of the natural strength of said network (100) in a short time in comparison with the prior art.
  • a method for biomodeling and bioprinting a self-supporting scaffold-free artificial hollow biological tissue network (100) is also proposed according to claim 8.
  • Said network (100) is for replacement of living tissue, and comprises continuous longitudinal multicellular aggregates (11) arranged in a plurality of bioprinted layers (22) which are located on top of one another, and wherein at least one of said bioprinted layers (22) is in shape of a closed loop such that a conduit for conveying gases and liquids is defined.
  • said method includes following steps:
  • a medical image which is preferably obtained using computer tomography (CT) or magnetic resonance imaging (MRI) is used to obtain geometric and topological information of targeted tissue (in this work, sample blood vessels, branched or unbranched).
  • CT computer tomography
  • MRI magnetic resonance imaging
  • STL messages model (2) of the targeted tissue is converted to a smooth parametric surface (5).
  • the computer model is then sliced for the layer-based 3D bioprinting process.
  • a novel self-supporting methodology is developed. Said self-supporting methodology is used to calculate corresponding locations for both longitudinal multicellular aggregates (11) and the support structures (12).
  • the bioprinting topology is generated in order to 3D bioprint the targeted tissue model directly from medical images, thus longitudinal multicellular aggregate (11) paths and support structure (12) paths are generated. Said longitudinal multicellular aggregate (11) and support structure (12) paths are used to control the bioprinter for 3D printing of a biomimetic network construct.
  • the details of the proposed methodology are given below.
  • a medical image of the original geometry of the targeted tissue must be captured and transformed into a computer aided model (CAD) of the tissue.
  • Three-dimensional medical images such as Magnetic Resonance Imaging (MRI) and Computer Tomography (CT) can be used for capturing the anatomically correct forms of internal organs and tissues.
  • MRI Magnetic Resonance Imaging
  • CT Computer Tomography
  • the main abdominal aorta model from a sample MRI data is used to highlight the proposed methods' capabilities for 3D bioprinting of macrovascular structures.
  • MRI or CT images are segmented using suitable software, e.g. the Mimics software.
  • a part of tissue e.g. abdominal aorta
  • a suitable imaging software was used for segmentation of the tissue.
  • the segmented region of the tissue is then converted into a 3D model.
  • Initial geometric information of the tissue structure is then represented as a computational model (2), preferably a mesh model or a stereolithography (STL) model.
  • the STL models are generated by tessellating the outer surface of the mesh model (2) with triangles.
  • STL models of targeted tissues here, sections of human blood vessels
  • Captured STL models of targeted tissues are not smooth and they could contain numerous polynomial (here, triangular) facets (8).
  • the resultant STL model of the targeted tissue requires to be represented by parametric surfaces.
  • a novel biomodeling method is developed to convert these mesh structures having triangular facets into smooth parametric surfaces suitable for 3D bioprinting.
  • the parametric representation of the targeted tissue model also eliminates any noise stemmed from the previous segmentation phase.
  • a representative drawing for understanding said biomodeling method of marching through section curves resulting of a centerline curve (10) of converting STL model to smooth parametric surfaces is shown in Figure 3
  • another representative drawing for further understanding said biomodeling method is shown in Figure 3 .
  • section curves (1) are generated from the mesh model (2). Center points (3) of each section curve (1) (contour) are then calculated. Generated center points (3) are used for approximation of a centerline curve (10). Then, smooth parametric surfaces (5) are generated along a trajectory of calculated centerline curve(s) (10).
  • Said curves are basically the starting and ending branched section curves of the STL model of a targeted tissue.
  • the bottom section curve (6) (EC b ) is used as the input.
  • section curve (1) which is a closed polyline curve
  • sections are section curves.
  • An algorithm is developed to compute the area weight-based center points (3) and respective radius values according to those vertices of the section curves (1).
  • the algorithm After several vertices (9) are marked as 'visited', the algorithm starts to march through the section curves starting from the bottom section curve (6) (EC b ) until it reaches to the top section curve(s) (7) (EC t ). If there are multiple closed section curves at a said section, the developed algorithm marks the previous section curve as starting point of said branched section curves. After one of the top section curves (7) is reached, the iteration continues from the starting point until reaching to all the top section curves (7).
  • Figure 2 (a) shows several section curves (1) and several center points (3) of section curves (1) for the tissue model. Furthermore, a section curves' maximally-inscribed sphere's radius will be the radius value for a corresponding center point (3).
  • the algorithm approximates a B-spline centerline curve (10) preferably using Cartesian coordinates of the center points (3). Then, the algorithm builds a B-spline curve, which will be the centerline curve (10) for the parametric surface (5).
  • the B-spline parametric surface (5) S k (u,v) of a tissue model is generated using said centerline curve(s) (10) for each branch (19) with respect to the average radius value of relevant center points (3) as shown in Figure 3 .
  • This operation basically sweeps a planar closed curve along a trajectory curve, as the centerline curve (10).
  • the trajectory centerline curve by CC b (u)
  • the planar closed curve by T(v).
  • M(u) is a 3 x 3 matrix incorporating rotation and non-uniform scaling of T(v) as a function of u.
  • a smooth tissue model (e.g. a blood vessel model) from the mesh model is determined using below algorithm.
  • Algorithm 1 Generating NURBS (non-uniform rational B-spline) Surface(s)
  • Algorithm 1 is very critical in biomimetical modeling of a targeted tissue model for 3D bioprinting topology calculation for both longitudinal multicellular aggregates ( 11) and support structures (12).
  • Figure 4 (a) shows several layers on said smooth surface (5) model where two of section curves (1) on said smooth surface (5) model are emphasized.
  • Longitudinal multicellular aggregate (11) is to be used layer-by-layer for obtaining targeted network (100) in accordance with said model (2).
  • S k (u,v) is generated as explained above, an optimum 3D bioprinting topology is to be determined.
  • Path planning for both longitudinal multicellular aggregate (11) and support structures (12) is calculated.
  • lengths of longitudinal multicellular aggregates (11) and support structures (12) to be used for constitution of each bioprinted layer (22) are calculated, such that said closed loops of longitudinal multicellular aggregates (11) and support structures (12) are without interruption.
  • the support structures (12) used in exemplary experiment according to the present invention was prepared according to the following sequence:
  • said aspiration is performed using an automated bioprinter, wherein said predetermined length of said capillary is equal to a respective length for a non-interrupted closed loop support structure (12) in a certain layer (22) to be bioprinted.
  • Both longitudinal multicellular aggregate (11) and support structures (12) are printed by a capillary (or respective capillaries), which capillary is preferably made of glass or another suitable bio-inert solid material, in a gel-like form layer by layer to form the 3D tissue network. Since longitudinal multicellular aggregates (11) are not self-shape preserving, both longitudinal multicellular aggregates (11) and support structures (12) should accordingly be placed on 'valleys' (13) of preceding layers as shown in Figure 5 in order to provide cell fusion and to print anatomically-correct model of the original tissue.
  • d capillary is the diameter of the capillary used.
  • N j,i (t) is unit normal vector on curve C j,0 (t) at a parameter t
  • Said number of longitudinal multicellular aggregates (11) for a calculative layer (14) i.e. on a bioprinted layer (22) is calculated by dividing the wall thickness of targeted tissue to the diameter of the longitudinal multicellular aggregate (11), and rounding the result up to nearest integer.
  • support structures (12) are printed first, and then longitudinal multicellular aggregate (11) is printed in order to prevent cell outflow and to preserve anatomically correct shape of the modeled tissue.
  • longitudinal multicellular aggregate (11) is printed in order to prevent cell outflow and to preserve anatomically correct shape of the modeled tissue.
  • a network (100) is composed of longitudinal multicellular aggregates (11) and support structures (12) that keep said longitudinal multicellular aggregates in the designed shape as intended.
  • each bioprinted layer (22) is sufficiently supported for stable cell aggregate printing as required, since the longitudinal multicellular aggregate (11) has less strength in comparison with an original biomaterial of the targeted tissue.
  • the term 'minSegmentLength' represents the length of a straight longitudinal piece (longitudinal multicellular aggregate or support structure) suitable to be bent or arched to form a closed loop without damaging said piece, and further suitable to form a pre-determined geometry for a certain function. If said piece is a longitudinal multicellular aggregate (11), said function can be maintaining a sufficient inner diameter allowing support structure to be placed; or if said piece is a support structure (12), said function can be approximately matching geometrical center points of both bases of longitudinal piece of support structure to form a proper closed loop for properly supporting an adjacent longitudinal multicellular aggregate loop (representing a section curve) of the same calculative layer (14).
  • Said 'suitability to be bent or arched without damaging a piece' depends on robustness (e.g. resilience) and shapes (i.e. geometries) of longitudinal pieces (11, 12) of longitudinal multicellular aggregate and support structure including their lengths and approximate diameters (or diameters for cylindrical pieces).
  • a suitable programming language e.g. Rhinoscript can be used for generating the above algorithms.
  • Initial geometries of targeted tissues are obtained by using suitable software e.g. Mimics and the patient's MRI data.
  • Algorithm 1 was used which extracts skeleton curves based on center lines (15) of the mesh model (2) and said algorithm generates smooth parametric surfaces (8) from said model (2).
  • path planning for both longitudinal multicellular aggregate (11) and support structures (12) is calculated.
  • Final outputs of the developed algorithms are 3D bioprinter instructions controlling the longitudinal multicellular aggregate (11) and support structure (12) heads to build the network (100) constructs layer by layer directly from the generated computer model.
  • the whole tissue network surface model which is extracted from an STL file obtained by Algorithm 1, was 50 mm high and its diameter is around 9 mm for the above explained experiment. In this step, dimensions of the network were exactly mimicked in bioprinting of both longitudinal multicellular aggregate (11) pieces and support structures (12).
  • a partial tissue model (for the examples presented in the figures, unbranched and branched human blood vessels) is extracted and its path planning for 3D bioprinting is obtained by Algorithm 2.
  • the developed methods according to the present invention are used for bio-printing of a network (100) comprising longitudinal multicellular aggregates (11) of a certain diameter (450 ⁇ m).
  • Longitudinal multicellular aggregates (11) and support structures (12) are 3D printed layer-by-layer according to the developed Self-Supporting method.
  • the longitudinal multicellular aggregates (11) are successfully printed at the valleys (13) formed by the support structure (11).
  • the 3D printed longitudinal multicellular aggregates (11) were perfectly supported by hydrogel.
  • novel computer aided algorithms are developed in order to 3D bioprint cell and support structures for scaffold-free tissue engineering e.g. macro-vascular tissue engineering.
  • the tool path design and 3D printing parameters are optimized for an anatomically correct 3D printing.
  • the captured geometry of said targeted tissue was converted to a computer-aided design model, i.e. model.
  • parametric surfaces are generated from said model.
  • a novel self-supporting methodology was developed with computational algorithms. Support structures and longitudinal multicellular aggregates are successfully printed with the developed self-supporting methodology.
  • the developed algorithms are implemented in a CAD software package, Rhino3D using Rhinoscript language.
  • the generated commands from the developed algorithms were used to control the bioprinter.
  • a small amount of appropriate medium is preferably dropped on printed longitudinal multicellular aggregate pieces during printing process.
  • the surface on which the network (100) is bioprinted is covered with a continuous support layer (17) which prevents longitudinal multicellular aggregate (11) from contacting said surface.
  • a covering support layer (16) is placed on bioprinted network (100).
  • said continuous support layer (17) and covering support layer (16) and support structures (12) comprise said appropriate medium to provide increased cell viability.
  • an automated dropping step of said appropriate medium is added to said method.
  • Modeled parts of human blood vessel constructs were successfully 3D bioprinted in accordance with the methods of the present invention.
  • a self-supporting scaffold-free artificial hollow biological tissue network for replacement of living tissue is provided. Furthermore, an artificial biological tissue network is obtained with a high reproducibility and without requiring any manual intervention. Said network is constructed in bottom-up direction. Additionally, said network achieves a natural mechanical strength in a shorter time with respect to the prior art. A method for obtaining such artificial biological tissue network is provided.

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Claims (9)

  1. - Réseau (100) pour le remplacement d'un tissu vivant, ledit réseau étant un réseau de tissu biologique creux artificiel sans échafaudage, comprenant une pluralité d'agrégats multicellulaires allongés (11) agencés dans une pluralité de couches bio-imprimées (22) qui sont situées les unes au-dessus des autres, comprenant en outre une surface interne (20) et une surface externe (21), dans lequel :
    - au moins l'une desdites couches bio-imprimées (22) a la forme d'une boucle fermée plane de telle sorte qu'un conduit de transport de fluides est défini, et
    - ledit agrégat multicellulaire allongé (11) est un mélange d'au moins deux types de cellules ;
    en outre dans lequel ledit réseau (100) est un conduit ramifié comprenant au moins deux ramifications (19) convergeant sur au moins un secteur dudit réseau (100) ;
    en outre dans lequel ledit agrégat multicellulaire allongé (11) comprend au moins un type de cellules choisi dans un groupe de types de cellules consistant en fibroblastes, cellules endothéliales et musculaires lisses.
  2. - Réseau (100) selon la revendication 1, dans lequel chaque boucle fermée dudit agrégat multicellulaire allongé (11) est en contact avec des structures de support biocompatibles (12) à la fois de ladite surface interne (20) et de ladite surface externe (21) pour servir de moule lors d'un processus de maturation du réseau de tissu biologique.
  3. - Réseau (100) selon l'une quelconque des revendications 1 ou 2, dans lequel ledit agrégat multicellulaire allongé (11) comprend en outre des cellules souches.
  4. - Réseau (100) selon l'une quelconque des revendications précédentes, dans lequel chaque agrégat multicellulaire allongé (11) est en contact direct de cellule à cellule avec au moins un agrégat multicellulaire allongé (11) adjacent.
  5. - Réseau (100) selon l'une quelconque des revendications précédentes, dans lequel lesdits agrégats multicellulaires allongés (11) sont complètement ou partiellement reliés les uns avec les autres.
  6. - Réseau (100) selon l'une quelconque des revendications précédentes, dans lequel ledit agrégat multicellulaire allongé (11) a un rapport d'aspect longueur/diamètre dans la plage de 20 à 250.
  7. - Réseau (100) selon l'une quelconque des revendications précédentes, dans lequel ledit agrégat multicellulaire allongé (11) a un diamètre dans la plage de 100 à 2500 micromètres.
  8. - Procédé de biomodélisation et de bio-impression d'un réseau (100) ramifié pour le remplacement d'un tissu vivant, ledit réseau étant un réseau de tissu biologique creux artificiel sans échafaudage, comprenant une pluralité d'agrégats multicellulaires allongés (11) agencés dans une pluralité de couches bio-imprimées (22) qui sont situées les unes au-dessus des autres, comprenant en outre une surface interne (20) et une surface externe (21), dans lequel au moins l'une desdites couches bio-imprimées (22) a la forme d'une boucle fermée plane de telle sorte qu'un conduit pour transporter des fluides est défini, et ledit agrégat multicellulaire allongé (11) est un mélange d'au moins deux types de cellules, dans lequel ledit agrégat multicellulaire allongé (11) comprend au moins un type de cellules choisi dans un groupe de types de cellules consistant en fibroblastes, cellules endothéliales et musculaires lisses ; ledit procédé comprenant les étapes suivantes :
    a) étapes de modélisation biomimétique pour l'obtention d'un modèle de surface lisse, répertoriées comme suit :
    - obtenir une série d'images médicales d'un tissu ciblé à bio-imprimer, ledit tissu ciblé étant un conduit ramifié comprenant au moins deux ramifications convergeant sur au moins un secteur dudit tissu ciblé ;
    - masquer et segmenter une région desdites images à l'aide d'un logiciel d'imagerie adapté ;
    - convertir ladite région segmentée en un modèle de maillage tridimensionnel (3D) (2) ayant des facettes polygonales (8) qui sont toutes également des parties de facettes adjacentes (8) d'un sommet (9) ;
    - définir des courbes de section de bord consistant en une courbe de section inférieure (6) et une ou plusieurs courbes de section supérieure (7) à utiliser comme courbes de section de début et de fin (1) du modèle, respectivement, une courbe de section (1) étant un réseau séquentiel de sommets (9) traités une fois jusqu'à ce qu'un sommet (9) soit traversé parce qu'il est déjà traité (c'est-à-dire que tout calcul est effectué pour ledit sommet (9)), et une section étant une couche calculatrice itérative (14) où une courbe de section respective (1) est calculée ;
    - sélectionner l'une desdites courbes de section inférieure (6) comme courbe de section de départ à utiliser comme entrée ;
    - marquer chaque sommet (9) dudit modèle (2) lorsque chaque sommet (9) est soumis à des calculs pour former une courbe de section (1) par rapport à une direction de marche desdits calculs ;
    - effectuer lesdits calculs à chaque sommet seulement une fois ;
    - obtenir une courbe à ligne brisée fermée en tant que courbe de section (1) en marquant chaque courbe de section (1) et un nombre de sommets (9) sur chaque courbe de section (1), de telle sorte que les calculs passent à une courbe de section non calculée adjacente (1) après avoir soumis chaque sommet (9) sur une courbe de section courante (1) à des calculs une fois ;
    - s'il existe plusieurs courbes de section (1) ayant entraîné le calcul d'une certaine section, définir la courbe de section précédemment calculée respective (1) comme point de départ d'une courbe de section de ramification ;
    - définir des ramifications (19) ayant le même nombre desdites multiples courbes de section (1) ;
    - définir un point central respectif (3) pour chaque courbe de section (1) reflétant des points centraux pondérés selon l'aire pour chaque courbe de section correspondante (1) ;
    - définir une valeur de rayon respective pour chaque courbe de section (1) reflétant un rayon d'une sphère inscrite de façon maximale de chaque courbe de section (1) ;
    - calculer les coordonnées d'un point central (3) pour chaque courbe de section (1) pour l'approximation d'une courbe médiane (10) pour chaque ramification (19) jusqu'à ce que leurs courbes de section supérieures respectives (7) soient atteintes ;
    - définir une courbe de section la plus haute (18) obtenue d'après un plus grand nombre de courbes de section (1) subséquentes calculées précédemment par comparaison du nombre de courbes de section (1) dans chaque ramification (19) ; définir la courbe de section supérieure (7) comme courbe de section la plus haute (18) s'il n'y a qu'une seule courbe de section (1) dans chaque couche calculatrice (14) ;
    - générer des surfaces paramétriques (5) avec des rayons calculés de courbes de section (1) le long d'une trajectoire d'une ou plusieurs courbes médianes respectives (10) pour la bio-impression 3D ;
    b) étapes de planification de chemin pour placer couche-par-couche lesdits agrégats multicellulaires allongés (11) et structures de support (12) sous la forme de pièces allongées constituées de matériau bio-inerte pour supporter ledit agrégat multicellulaire allongé (11) sur une surface de substrat biocompatible horizontale à travers des structures de support respectives (12), à partir à la fois d'une surface interne (20) et d'une surface externe (21) dudit réseau (100), lesquelles structures de support (12) moulent ledit réseau (100) au cours d'un processus de maturation :
    - calculer un nombre total de couches bio-imprimées (22) par division de la distance entre ladite courbe de section la plus haute (18) et ladite courbe inférieure (6) de ladite surface paramétrique (5) par le diamètre d'un agrégat multicellulaire allongé respectif (11) ;
    - couper ladite surface paramétrique (5) avec des couches calculatrices successives (14) et obtenir des courbes de contour respectives pour chaque couche calculatrice (14) ;
    - décaler chaque courbe de contour avec la grandeur du diamètre de l'agrégat multicellulaire allongé (11) jusqu'à atteindre une épaisseur de réseau ciblé (100) aux couches bio-imprimées respectives (22), de telle sorte que le diamètre le plus interne de chaque couche bio-imprimée (22) n'est pas inférieur à un diamètre interne correspondant du réseau ciblé (100) aux couches bio-imprimées respectives (22) ;
    - déterminer un nombre de couches de structure de support en boucle fermée qui doivent supporter ladite courbe supérieure (7) de façon périphérique à partir à la fois de l'intérieur (20) et de l'extérieur (21) de ladite courbe supérieure (7) ;
    - pour chaque couche bio-imprimée (22), calculer un nombre pour des couches de structure de support extérieures (23) recouvrant/supportant les courbes précédemment calculées d'une couche supérieure adjacente de façon périphérique à partir de l'extérieur (21), de telle sorte que ledit nombre est obtenu par addition itérative avec au moins '1' pour chaque couche en partant de ladite couche supérieure (7) jusqu'à ladite couche inférieure (6), et avec une valeur de départ égale au nombre déterminé ci-dessus de couches de structure de support extérieures (23) supportant ledit réseau en boucle fermée (100) à partir l'extérieur (21) ;
    - pour chaque couche, calculer un nombre de couches de structure de support intérieures (24) recouvrant/supportant les courbes précédemment calculées de ladite couche supérieure adjacente de façon périphérique à partir de l'intérieur (20), de telle sorte que ledit nombre est obtenu par addition itérative avec '1' pour chaque couche en partant de ladite couche supérieure (7) jusqu'à ladite couche inférieure (6), et de telle sorte que l'addition itérative avec '0' au lieu de '1' est appliquée si un nombre égal à la plus grande valeur entière de division d'un rayon interne correspondant pour une courbe de section (1) par le rayon de la structure de support (12) est atteinte pour une couche ;
    - si de multiples structures de support (12) se croisent de façon adjacente à une même couche bio-imprimée (22), former une structure de support en boucle fermée à ladite couche par réunion desdites multiples structures de support (12) ;
    - déterminer des longueurs pour des agrégats multicellulaires allongés (11) et des structures de support (12) à aspirer dans des capillaires et à placer pour construire chaque couche bio-imprimée respective (22) ;
    - déterminer des coordonnées de dépôt des structures de support (12) et des agrégats multicellulaires allongés (11) conformément aux étapes ci-dessus ;
    - générer et enregistrer des commandes séquentielles pour la commande d'une bio-imprimante pour le dépôt couche-par-couche de structures de support (12) puis d'agrégats multicellulaires allongés (11) en partant de ladite couche inférieure (6), jusqu'à ladite couche la plus haute (7, 18) ;
    - envoyer lesdites commandes à une bio-imprimante.
  9. - Procédé selon la revendication 8, dans lequel l'aspiration et la bio-impression sont effectuées automatiquement par une bio-imprimante automatisée comprenant ledit capillaire.
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Families Citing this family (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9815268B2 (en) * 2013-03-22 2017-11-14 Markforged, Inc. Multiaxis fiber reinforcement for 3D printing
US10259160B2 (en) 2013-03-22 2019-04-16 Markforged, Inc. Wear resistance in 3D printing of composites
US11981069B2 (en) 2013-03-22 2024-05-14 Markforged, Inc. Three dimensional printing of composite reinforced structures
US9956725B2 (en) 2013-03-22 2018-05-01 Markforged, Inc. Three dimensional printer for fiber reinforced composite filament fabrication
US9186848B2 (en) 2013-03-22 2015-11-17 Markforged, Inc. Three dimensional printing of composite reinforced structures
US9186846B1 (en) 2013-03-22 2015-11-17 Markforged, Inc. Methods for composite filament threading in three dimensional printing
US10682844B2 (en) 2013-03-22 2020-06-16 Markforged, Inc. Embedding 3D printed fiber reinforcement in molded articles
US9126365B1 (en) 2013-03-22 2015-09-08 Markforged, Inc. Methods for composite filament fabrication in three dimensional printing
US9156205B2 (en) 2013-03-22 2015-10-13 Markforged, Inc. Three dimensional printer with composite filament fabrication
US10953609B1 (en) 2013-03-22 2021-03-23 Markforged, Inc. Scanning print bed and part height in 3D printing
CN107813495A (zh) 2013-03-22 2018-03-20 格雷戈里·托马斯·马克 三维打印
US9149988B2 (en) 2013-03-22 2015-10-06 Markforged, Inc. Three dimensional printing
US11237542B2 (en) 2013-03-22 2022-02-01 Markforged, Inc. Composite filament 3D printing using complementary reinforcement formations
US9688028B2 (en) 2013-03-22 2017-06-27 Markforged, Inc. Multilayer fiber reinforcement design for 3D printing
US9579851B2 (en) 2013-03-22 2017-02-28 Markforged, Inc. Apparatus for fiber reinforced additive manufacturing
US9694544B2 (en) 2013-03-22 2017-07-04 Markforged, Inc. Methods for fiber reinforced additive manufacturing
WO2014197732A2 (fr) 2013-06-05 2014-12-11 Markforged, Inc. Procédés de fabrication d'additif renforcé de fibres
WO2016154070A1 (fr) 2015-03-20 2016-09-29 William Marsh Rice University Bioimpression hypothermique en 3d de tissus vivants supportée par un système vasculaire pouvant être perfusé
US10751943B2 (en) * 2015-08-24 2020-08-25 Siemens Healthcare Gmbh Personalized creation from medical imaging
US9824492B2 (en) 2016-03-24 2017-11-21 Vital Images, Inc. Hollow object model visualization in medical images
EP3471789B1 (fr) * 2016-06-16 2025-07-09 Aspect Biosystems Ltd. Implant méniscal bio-imprimé et ses procédés d'utilisation
US11254901B2 (en) 2016-07-12 2022-02-22 Deka Products Limited Partnership System and method for printing tissue
US10345208B2 (en) 2016-07-12 2019-07-09 Deka Products Limited Partnership System and method for applying force to a device
US11299705B2 (en) 2016-11-07 2022-04-12 Deka Products Limited Partnership System and method for creating tissue
US12297416B2 (en) 2017-07-12 2025-05-13 Deka Products Limited Partneship System and method for transferring tissue
US10570362B2 (en) 2017-07-12 2020-02-25 Deka Products Limited Partnership System and method for transferring tissue
EP3727484A1 (fr) 2017-12-20 2020-10-28 Aspect Biosystems Ltd. Implant méniscal bio-imprimé et ses procédés d'utilisation
EP4653192A2 (fr) * 2019-04-26 2025-11-26 William Marsh Rice University Réseaux multivasculaires et topologies intravasculaires fonctionnelles dans des hydrogels biocompatibles
CN112057208B (zh) * 2019-06-11 2024-12-10 江苏双恩智能科技有限公司 一种人工关节涂层结构及其3d打印数据模型生成方法
WO2022126594A1 (fr) * 2020-12-18 2022-06-23 中国科学院深圳先进技术研究院 Procédé de construction d'échafaudage fractal pour l'ingénierie tissulaire osseuse
CN114949369B (zh) * 2021-02-19 2023-09-05 清华大学 一种人工组织器官的封装装置及其制备方法和应用
US12030256B2 (en) 2021-02-19 2024-07-09 The Board Of Trustees Of The University Of Illinois Coated tubular construct for biological and industrial applications and method of making a tubular construct
CN114274505B (zh) * 2021-12-23 2022-08-30 山东大学 一种三明治板熔融沉积打印支撑结构生成方法及系统
TWI848552B (zh) * 2023-02-09 2024-07-11 中國醫藥大學 多孔單元體結構導入實體模型的方法
CN116212109A (zh) * 2023-03-07 2023-06-06 中国科学技术大学 一种3d打印生物墨水、其制备方法、3d打印活体功能材料及其制备方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103767804A (zh) * 2014-01-20 2014-05-07 清华大学 一种具有微流体通道的血管化组织结构及其制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005081970A2 (fr) 2004-02-24 2005-09-09 The Curators Of The University Of Missouri Agregats cellulaires d'auto-assemblage et procedes de fabrication de tissu genetiquement modifies utilisant de tels agregats
JP5624981B2 (ja) 2008-06-24 2014-11-12 ザ・キュレーターズ・オブ・ザ・ユニバーシティ・オブ・ミズーリＴｈｅ Ｃｕｒａｔｏｒｓ Ｏｆｔｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｍｉｓｓｏｕｒｉ 自己集合性多細胞体、および前記多細胞体を用いて３次元の生物構造体を作製する方法
GB2478801B (en) * 2010-03-16 2012-05-30 Organovo Inc Multilayered vascular tubes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103767804A (zh) * 2014-01-20 2014-05-07 清华大学 一种具有微流体通道的血管化组织结构及其制备方法

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